Bile acids epimers and side chain homologues are present in the human colon. To test whether such secondary bile acids possess secretory activity, cultured T₈₄ colonic epithelial cells were used to quantify the secretory properties of synthetic epimers and homologues of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). Methods: Cl^- secretion was measured as changes in short circuit current (I_sc, in µA/cm²) using voltage-clamped monolayers of T₈₄ cells mounted in Ussing chambers. Bile acids were added at 0.5 mM, a concentration that did not alter transepithelial resistance. Data were expressed as peak I_sc (mean ± standard deviation). Results: When added bilaterally, DCA stimulated a I_sc response of 15.7 ± 12.5. The 12-OH epimer of DCA was less potent (I_sc = 8.0 ± 1.7), whereas its 3-OH epimer had no effect. CDCA stimulated secretion (I_sc = 8.2 ± 5.5), whereas both its 7-OH and 3-OH epimers were inactive, as was lithocholic acid. Homo-DCA, (one additional side chain carbon) was active (I_sc = 7.8 ±4.8) whereas norDCA (one fewer carbon) and dinorDCA (two fewer carbons) were not. Taurine conjugates of DCA and CDCA stimulated secretion (I_sc = 12.3 ± 7.5 and 8.8 ± 4.8, respectively) from the basolateral side but not the apical side. Uptake of taurine conjugates from the basolateral but not the apical side was shown by mass spectrometry. Conclusions: These studies indicate marked structural specificity for bile acid-induced Cl^- secretion and show that modification of bile acid structure by colonic bacteria modulates the secretory properties of these endogenous secretagogues.